Project Summary/Abstract The cytoskeleton consists of several filamentous proteins including actin filaments and microtubules, that regulate nearly all vital cell processes including: cell division, morphogenesis, phagocytosis, and movement/motility. Exactly how the activities of actin and microtubules are coordinated to carry out these cell processes is not fully understood. The goal of this research is to determine how the dynamics of the actin and microtubules are coordinated in two ways: 1) by a group of regulatory proteins that bind to actin and MTs and also interact with each other: CLIP-170, mDia1, EB1, and IQGAP1; and 2) through the formation of disease- relevant protein aggregates (liquid droplets). This project uses classical biochemistry and cell biology assays combined with a unique 4-color advanced microscopy imaging system that permits the simultaneous monitoring of purified actin and microtubules with fluorescently labeled single molecules of interacting proteins to illuminate detailed molecular mechanisms. We will further test the physiological and disease-relevance of these mechanisms during critical cell processes (i.e. cell division and migration) in several cell types. This research will advance a dynamic and emerging field by defining complex molecular interactions and mechanisms of microtubule-actin crosstalk that underlie a host of fundamental biological processes and neurological disease.